Rubella virus is a major human pathogen. Infection with rubella virus can cause serious birth defects and chronic disease. There was a mini-epidemic of both rubella and congenital rubella syndrome in the United States between 1989 and 1991.
Rubella was first described in the eighteenth century in Germany. The symptoms of a rash and mild fever were similar to those of measles, so the disease was given the name German measles. The name “rubella” was coined in 1814 when physicians realized that the disease was unique and was not merely a variant of scarlatina (scarlet fever) or rubeola (measles).
Rubella is a relatively harmless disease in young children. However, during the first trimester of pregnancy, rubella virus infection can cause fetal death. If the fetus survives, it may be born deaf or have cataracts, cardiac abnormalities, microcephaly, motor deficits or other congenital anomalies. The infant may also be born with thrombocytopenic purpura, hepatosplenomegaly, icterus, anemia, and low birth weight. The presence of one or more of these defects has been termed “congenital rubella syndrome” or CRS.
The rubella virus was isolated in 1962 at the beginning of a worldwide rubella epidemic which lasted from 1962 to 1965. This epidemic peaked in the United States in 1964, resulting in the birth of approximately 20,000 infants exhibiting CRS.
Scientists began development of an effective vaccine against the rubella virus during the rubella epidemic. Effective attenuated vaccines became available in the late 1960's and are still used today. These attenuated vaccines are live viruses that have been passaged to reduce their virulence. Attenuated vaccines produce immunity, but can cause disease. Protection is believed to persist for at least 15 years after inoculation with the attenuated rubella vaccine.
Various vaccination schedules have been set up in different parts of the world to eliminate rubella infection, especially of the human fetus. The rubella immunization program established in Great Britain requires vaccination of all girls between the ages of 10 and 14. The United States immunization program vaccinates infants at approximately 15 months and requires a certificate of vaccination prior to attending school. The United States program is designed to eradicate the disease among the population that is most responsible for transmission of rubella, whereas the program of Great Britain seeks to achieve complete protection for those at risk for pregnancy. One disadvantage to the United States program is that protection against rubella may dissipate at the very time when immunity is most needed, namely, during the child-bearing years.
Vaccination of women of child-bearing age having undetectable antibody titers is recommended in both the United States and Great Britain. However, there are several risks to this procedure. First, there is a risk that these women may be pregnant and not be aware of their pregnancy, or they may become pregnant within a few months following immunization. Vaccination against rubella is contraindicated in pregnant women because the live virus in the vaccine can cross the placenta and infect the fetus. Pregnant women who have not previously been infected with the rubella virus or who have not been vaccinated prior to becoming pregnant are advised to refrain from becoming vaccinated during their pregnancy. These women are therefore at risk for contracting rubella by coming in contact with infectious persons, including those recently vaccinated with the attenuated vaccine.
Vaccination of older women has been associated with chronic arthritis and neurological symptoms. Scientists believe that these symptoms may be due to the persistent nature of the attenuated rubella virus in the currently available vaccines.
Rubella virus is a small, quasi-spherical, enveloped, nonsegmented, plus-strand RNA virus that is the the sole member of the rubivirus genus of the togavirus family (Togaviridae). Molecular biology of rubella virus is summarized by Frey, T. K. in Adv. Virus Res. 44:69-160 (1994). One other member of the togavirus family is alphaviruses (see Strauss, J. H., and E. G. Strauss, Microbiol. Rev. 58:491-562. (1994) for a detailed description), which include a number of viruses pathogenic for vertebrates, including humans. The rubella virion (virus particle) consists of single-stranded RNA encapsidated in an icosahedral nucleocapsid surrounded by a lipid envelope. Multiple copies of a viral protein, designated the C protein (MW (molecular weight)=32,000-38,000 daltons), make up the nucleocapsid. Two types of viral glycoprotein, designated E1 and E2 (MW=53,000-58,000 daltons and 42,000-48,000 daltons, respectively), are embedded in the envelope, as reported by Waxham, M. N. and Wolinsky, J. S., Virology 126:194-203 (1983). The E2 glycoprotein has been further subdivided into two subgroups, designated E2a and E2b, by their ability to migrate differently when resolved by polyacrylamide gel electrophoresis, as described by Oker-Blom, C., et al., J. Virol. 46:964-973 (1983). E1 is the viral hemagglutinin. Neutralizing epitopes have been found on both E1 and E2 by Waxham, M. N. and Wolinsky, J. S., Virology 143:153-165 (1985) and Green, K. Y., and Dorsett, P. H., J. Virol., 57:893-898 (1986).
The rubella genome consists of RNA of positive polarity that is roughly 10,000 nucleotides long and is capped and polyadenylated. In infected cells, three viral RNA species are synthesized: the genomic RNA, which also is the mRNA for translation of the nonstructural proteins (whose function is in viral RNA synthesis) from a long open reading frame (ORF) at the 5′ end of the genome; a complementary genome-length RNA of minus polarity which is the template for synthesis of plus-strand RNA species; and a subgenomic (SG) RNA which is initiated internally and contains the sequences of the 3′-terminal one-third of the genome (3327 nucleotides) and serves as the mRNA for the translation of the structural proteins. The structural proteins are proteolytically processed from a polyprotein precursor during translation. The order of these proteins in the polyprotein is NH2—C-E2-E1—COOH, as reported by Oker-Blom, C., et al. (1983); Oker-Blom, C., J. Virol. 51:354-358 (1984). In the other togavirus genus, the alphaviruses, synthesis of the SG RNA is directed by a short, approximately 25 nucleotide sequence located immediately upstream from the SG start site known as the SG promoter, as reported by Strauss, et al., Microbiol. Rev. 58:491-562 (1994). The exact location of the SG promoter in rubella virus genome is not known.
Recombinant vaccines are based on live microorganisms which have been genetically manipulated so that they are not pathogenic, but result in immunity against the virulent organism. Recombinant vaccines can only cause disease if a rare genetic mutation or recombinant event occurs which allows the microorganism to revert to wild type. A recombinant vaccine is generally safer and more effective than an attenuated vaccine because the engineered mutations remove or inactivate only specific portions of the genome, whereas attenuated vaccines contain random mutations. In order to develop a recombinant vaccine, one must first have the nucleic acid sequence of the entire viral genome, including both the information required for infection and at least limited replication of the virus, and for antigenicity. Once the entire sequence has been determined, a cDNA clone can be produced that is infectious and can be modified to be non-virulent.
An infectious cDNA clone is a complete DNA copy of an RNA virus genome contained in a vector, such as a plasmid, from which RNA transcripts of the genome can be synthesized in vitro. In the case of positive-polarity RNA viruses such as rubella, such transcripts are infectious when transfected into cells. The development of an infectious clone is a landmark event in the molecular biology of any RNA virus. Although an infectious clone for rubella virus has been described (Wang, et al., J. Virol. 68:3550-3557 (1994)), this cDNA clone displayed low infectivity (approximately 5 plaques/10 μg of transcripts). Increasing the infectivity of this clone would increase the efficiency of a recombinant attenuated rubella vaccine derived from the clone and would provide an improved molecular biology tool for studying rubella virus replication.
However, successful generation of highly infectious cDNA clones has often been problematic due to the presence of mutations in the virus RNA template population caused by the inherent mutability of RNA viruses, the relatively low fidelity of the DNA polymerases used in cDNA synthesis, instability and toxicity of viral sequences in bacterial hosts, and the infidelity of the RNA polymerases used for in vitro transcriptions (Boyer and Haenni, Virology 198:415-426 (1994)). It is clear that there remains a strong need for an infectious cDNA clone of the rubella virus genome having a higher infectivity than currently available rubella virus clones, and for a recombinant rubella virus vaccine. The isolation of a highly infectious cDNA clone will be useful for the development of a recombinant rubella vaccine vector. A recombinant vaccine vector based on live, attenuated rubella vaccines is also highly desirable in a pediatric setting, where immunization with a recombinant rubella vaccine expression vector can be used to induce immunity against rubella alone, or both rubella and another virus or viruses whose genes may be introduced into the vector. Such vaccine vector is also desirable in an adult patient setting, where a recombinant rubella vaccine is needed that can be safely administered to pregnant and older women without risk of birth defects, auto immune disease, or neurologic symptoms. Thus, rubella virus expression vectors that can be used to produce and develop recombinant vaccines against rubella and other viruses would be highly useful to combat rubella and other diseases in various populations. Also, the development of a method to improve the stability of togavirus expression vectors would be very useful for the development of the expression vectors and recombinant vaccines for rubella virus and other viruses of this family, such as alphaviruses. The instability of alphavirus expression vectors is well known, as reported by Pugachev, K. V., et al., Virology 209:155-166 (1995) and Pugachev, K. V., et al., Virology 212:587-594 (1995). Futhermore, rubella virus expression vectors would serve as valuable molecular biology tools to study rubella virus and other viruses of the togavirus family.